Cloning of QaGDU3 gene from Quercus aquifolioides Rehd. et Wils. and its genetic transformation into Arabidopsis thaliana (L.) Heynh.
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摘要: 以川滇高山栎(Quercus aquifolioides Rehd. et Wils.)叶片为材料，分别采用不依赖于连接反应的克隆法、农杆菌花序侵染、实时荧光定量PCR、图像分析等方法克隆了QaGDU3基因，获得QaGDU3转基因拟南芥(Arabidopsis thaliana(L.)Heynh.)并分析转基因植物的表型变化，利用生物信息学对QaGDU3蛋白与其他物种的GDU3蛋白序列间的差异和进化关系进行了分析。结果显示，GDU3是种子植物特有的基因；QaGDU3属于阴离子渗透酶ArsB/NhaD超家族成员，可能具有氨基酸转运功能。QaGDU3基因在拟南芥中过表达能够激活水杨酸(SA)途径中AtPR1、AtACD6、AtCBP60g和AtPAD4基因的表达，且转基因拟南芥莲座大小随着QaGDU3基因表达量升高而变小。研究结果说明GDU3是种子植物所特有的氨基酸转运蛋白并参与植物生长发育的调控。Abstract: We cloned the QaGDU3 gene from Quercus aquifolioides Rehd. et Wils. and obtained transgenic Arabidopsis thaliana (L.) Heynh. using ligation-independent cloning (LIC) and floral dipping transformation. We evaluated QaGDU3 expression and analyzed phenotypic changes in transgenic plants using quantitative real-time polymerase chain reaction (qRT-PCR) and image analysis, respectively. We investigated the structure, sequence divergence, and evolutionary relationship of QaGDU3 with related species. Structural analysis showed that QaGDU3 is an acidic and unstable hydrophilic transmembrane protein. Phylogenetic analysis indicated that GDU3 may only be present in seed plants. The qRT-PCR results showed that overexpression of the QaGDU3 gene in transgenic plants activated the expression of the AtPR1 (PATHOGENESIS-RELATED GENE 1), AtACD6 (ACCELERATED CELL DEATH 6), AtCBP60g (CAM-BINDING PROTEIN 60-LIKE g), and AtPAD4 (PHYTOALEXIN DEFICIENT 4) genes involved in the salicylic acid (SA) pathway. Furthermore, the rosette size of transgenic A. thaliana decreased with the increase in QaGDU3 expression. These data suggest that GDU3 may be a seed plant-specific amino acid transporter and that overexpression of QaGDU3 can affect plant growth and development.
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Keywords:
- Quercus aquifolioides /
- Sequence analysis /
- Gene function
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